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Développement d'une nouvelle forme galénique pour l'administration intraoculaire de rifampicine / Development of rifampicin encapsulated microparticles for intraocular injectionLee, Mi-Yeon 08 June 2011 (has links)
Des microparticules de rifampicine pour l'administration intraoculaire ont été développées en vue du traitement prophylactique de l'endophtalmie post-chirurgicale. Ces microparticules à base d'un polymère biodégradable, le PLGA, sont produites par émulsion-diffusion. Tout d'abord l'influence des paramètres de formulation et de procédé sur les caractéristiques des microparticules (taille, charge, taux d'encapsulation…) a été étudiée. La formulation a par la suite pu être optimisée par la mise en place d'un plan d'expérience, ce qui a permis de mettre en valeur les paramètres clefs influençant les propriétés des microparticules (concentrations en PLGA et PVA, présence ou non d'un coeur huileux…) et donc d'optimiser la formulation pour obtenir des microparticules adéquates pour l'administration intraoculaire de rifampicine (1μm<taille<10μm, encapsulation maximale…). Les études de libération de la rifampicine in vitro, dans des conditions mimant celles physiologiques, ont montré que d'une part plus de 50% de la rifampicine était libérée en 1h permettant d'obtenir une dose "d'attaque" et d'autre part que les microparticules permettaient de maintenir une libération prolongée sur 24h avec une concentration > CMI50 de Staphylococcus epidermidis (pathogène modèle utilisé pour les études microbiologiques). Enfin, une évaluation de l'effet bactéricide et antiadhérent sur lentilles intraoculaires (IOLs) de la rifampicine microencapsulée a été menée en présence de S. epidermidis. Les microparticules de rifampicine présentent une efficacité nettement supérieure à la solution contrôle de rifampicine puisque une bactéricidie totale a pu être observée 30h après injection (contre 103 CFU/mL restantes avec la solution contrôle), avec une activité antiadhérente nettement plus marquée (aucune bactérie détectée sur IOLs après 18h). Les microparticules de rifampicine apparaissent donc comme système d'administration intraoculaire prometteur dans le traitement prophylactique de l'endophtalmie post-opératoire / In this study, rifampicin-loaded microparticles were designed for intra ocular injection after cataract surgery to prevent postoperative endophthalmitis. Microparticles were produced by emulsification diffusion method using PLGA as biodegradable polymer and PVA as stabilizer agent. Influence of formulation and process parameters on microparticle characteristics (size, zeta potential, encapsulation efficiency…) was firstly investigated. Main parameters influencing the properties of particles were then identified by experimental design and formulation parameters were optimized to get desired particle sizes for intraocular delivery (between 1 and 10 μm) and the highest encapsulation of rifampicin. In vitro release studies of rifampicin in BSS at 37°C were performed showing firstly a burst release in 1 hour (more than 50% of rifampicin released), followed by a sustained release with a rifampicin concentration in the medium higher than the ICM50 on Staphylococcus epidermidis during 24h. Finally, the antimicrobial efficiency of rifampicin microparticles was evaluated on S. epidermidis. The results showed a higher bactericidal effect of encapsulated rifampicin than the control rifampicin solution; no bacteria were retrieved after 30h in the medium. Moreover, the antiadhesive property of rifampicin microparticles on intraocular lens (IOLs) was demonstrated since no bacteria were found on IOLs after 18h of incubation. From these experimental results, rifampicin encapsulated polymeric microparticles seem to be a good candidate for intraocular delivery of rifampicin in postoperative endophthalmitis prophylaxis
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Efeitos antagônicos da prostaglandina D2 e prostaglandina E2 na resposta imune durante infecção experimental por Histoplasma capsulatum / Opposite effects of prostaglandin D2 and prostaglandin E2 in immune response during experimental infection by Histoplasma capsulatum.Pereira, Priscilla Aparecida Tartari 30 October 2013 (has links)
O Histoplasma capsulatum é um fungo dimórfico, patogênico e responsável por graves lesões pulmonares. A infecção é adquirida pela inalação de conídios e posterior conversão para leveduras nos alvéolos e bronquíolos, onde são fagocitadas por macrófagos alveolares residentes e leucócitos que migram para o local da infecção. Recentemente, demonstramos que animais infectados com H. capsulatum e tratados com inibidor da síntese de prostaglandinas apresentaram diminuição de carga fúngica nos pulmões e baço, aumento da produção de nitrito e da fagocitose de leveduras por macrófagos alveolares, e maior sobrevivência, quando comparados com os animais somente infectados. Porém, neste estudo não foram determinados quais subtipos de prostaglandinas participam na patogênese da histoplasmose. Vários grupos de pesquisa têm demonstrado que PGD2 e PGE2 podem ter ações biológicas distintas quanto à remoção de microrganismos no hospedeiro. Desta maneira, é fundamental o entendimento do papel da PGD2 e da PGE2 nos mecanismos efetores dos macrófagos na defesa do hospedeiro, especialmente na histoplasmose. Portanto, o objetivo deste estudo foi investigar a participação da PGD2 e PGE2 na infecção experimental por H. capsulatum. Assim, demonstramos que a PGD2 aumentou a fagocitose e mecanismos microbicidas de macrófagos alveolares infectados in vitro com H. capsulatum. Observamos ainda que a 15dPGJ2, metabólito da PGD2, aumentou somente a fagocitose, e PGE2 inibiu os mecanismos efetores do macrófago. Mostramos ainda o aumento de BLT1 em macrófagos alveolares após adição de PGD2, e a possível ligação desta ao BLT1, e de LTB4 em DP2. Além disso, caracterizamos micropartículas de PLGA contendo PGD2 (MS-PGD2), e investigamos seus efeitos. O tamanho, carga elétrica e morfologia das micropartículas foram adequados para um tratamento intranasal e para fagocitose por macrófagos alveolares. As MS-PGD2 foram fagocitadas e capazes de ativar NF-B, e consequentemente, influenciar na produção de nitrito, IL-1, TNF-, IL-6 e TGF-. Com base nestes dados, avaliamos os efeitos do tratamento da MS-PGD2 ou da MS-PGE2 em animais infectados com H. capsulatum. Estas foram administradas via intranasal em animais infectados e tratados ou não com celecoxibe. Verificamos a diminuição da carga fúngica nos pulmões e baço, diminuição do infiltrador celular no espaço broncoalveolar e de citocinas inflamatórias no pulmão após tratamento com MS-PGD2. Contrariamente, após tratamento da MS-PGE2 observamos maior carga fúngica nos pulmões e baço, e aumento da inflamação no tecido e maior produção de IL-10. Além disso, demonstramos que no 21° dia após infecção, referente ao 7° dia após o término do tratamento com MS-PGD2, a carga fúngica manteve-se reduzida nos pulmões, comprovando assim a eficácia deste tratamento. Posteriormente, utilizando inibidores específicos, HQL-79 e CAY10526, mostramos respectivamente o papel protetor da PGD2 e o deletério da PGE2 na histoplasmose. Em conjunto, nossos dados contribuíram para o entendimento das funções antagônicas da PGD2 e PGE2 nesta micose. / Histoplasma capsulatum is a pathogenic dimorphic fungus and responsible for severe pulmonary lesions. Infection is acquired by inhalation of conidia and posterior conversion to yeasts in the alveoli and bronchioles, in which they are phagocyted by resident alveolar macrophages and leukocytes that migrate to the local infection. Recently, we demonstrate that mice infected by H. capsulatum and treated with inhibitor of prostaglandins synthesis presented a decrease in fungal burden in lungs and spleen, increase in nitrite production and uptake of yeasts by alveolar macrophages, and more survival, when compared with animals only infected. However, in this study, it was not determined what subtypes of prostaglandins participate in pathogenesis of histoplasmosis. Many research groups have demonstrated that PGD2 and PGE2 can have different biological effects regarding to microorganisms elimination in the host. Thus, it is primordial the understanding about the role of PGD2 and PGE2 on effector mechanisms of macrophages in host defense, especially in histoplasmosis. Therefore, the aim of this study was to investigate the role of PGD2 and PGE2 on experimental infection by H. capsulatum. So, we verify that PGD2 increased the uptake and microbicidal mechanisms of alveolar macrophages infected in vitro by H. capsulatum. 15dPGJ2, a PGD2 metabolite, increased only the phagocytosis, and PGE2 inhibited the effector mechanisms of macrophages. Among these results, we showed an increase of BLT1 expression on alveolar macrophages after addition of PGD2, and a possible binding of this mediator to BLT1, and of LTB4 to DP2. Later, as tool of therapeutic investigation, we used PGD2 encapsulation in biodegradable polymer, PLGA, in order to preserve its stability. Size, zeta potential and morphology were adequate for a possible intranasal treatment and uptake by alveolar macrophages. MS-PGD2 were phagocyted and able to activate NF-B, and consequently, to modulate nitrite, IL-1, TNF-, IL-6 and TGF- production. In this context, we purpose a treatment of the infection with MS-PGD2, in comparison to treatment with PGE2. MS-PGD2 were administrated via intranasal in infected mice, treated or not with celecoxib. We verify a decrease of fungal burden in lungs and spleen, less cellular infiltrate and decrease of some inflammatory cytokines. In contrast, after treatment of MS-PGE2, we observed greater fungal burden in the lungs and spleen, and an increase of the tissue inflammation and production of IL-10. Furthermore, we show that on day 21 after infection, referring to the 7th day after the treatment with MS-PGD2, fungal burden remained reduced in the lungs, thus proving the effectiveness of the treatment. Subsequently, using specific inhibitors, HQL-79 and CAY10526, respectively show the protective role of PGD2 and in deleterious to PGE2 in histoplasmosis. Together, our data contribute to the understanding of the antagonistic functions of PGD2 and PGE2 in this mycosis.
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Avaliação da influência da lipoxina A4 encapsulada em micropartículas de PLGA na cicatrização de úlceras cutâneas em ratos / Influence of Lipoxin A4 encapsulated in PLGA microparticles on wound healing in ratsReis, Mouzarllem Barros dos 27 June 2016 (has links)
Lipoxina A4 (LXA4) é um eicosanoide derivado do metabolismo do ácido araquidônico pelas lipoxigenases (5, 12 e 15-LO), tendo propriedades antiinflamatórias e pró-resolução. A estratégia de uso de LXA4 encapsulada em micropartículas de PLGA como fármaco tem fundamento nas propriedades que tais polímeros têm de preservar as atividades biológicas de moléculas diversas, como dos lipídeos, e promover liberação prolongada e sustentada das mesmas. No presente estudo, tivemos como hipótese de trabalho que a encapsulação de LXA4 em micropartículas de PLGA (LXA4-MS) preserva suas atividades biológicas, e mais eficientemente, acelera o fechamento de úlceras induzidas na pele de ratos. Para tanto, um modelo de úlceras cutâneas na região dorsal de ratos foi utilizado. As LXA4-MS foram fixadas em pele com adesivo biológico de fibrina, e seus efeitos comparados com aqueles induzidos por micropartículas vazias (Un-MS), LXA4 solúvel ou veículo (PBS). Nossos resultados mostraram que LXA4-MS aceleram a cicatrização da ferida, pois no 7º dia após a lesão, reduziu em 80% o diâmetro da úlcera inicial, enquanto no mesmo período, nas úlceras tratadas com LXA4 solúvel, Un-MS ou veículo ocorreu diminuição de somente 60%, 45% e 39%, respectivamente. O aumento do índice de cicatrização induzido pelo tratamento das úlceras com LXA4-MS foi acompanhado pela diminuição das citocinas pró-inflamatórias IL- 1?, TNF-?, e aumento de TGF- ?, uma citocina antiinflamatória indutora da deposição de colágeno. Além disso, nas úlceras tratadas com LXA4-MS o infiltrado inflamatório foi reduzido no tecido de cicatrização, como demonstrado pela diminuição de infiltrado celular, de MPO e de mRNA da metaloproteinase MMP8. Por outro lado, LXA4-MS induziu aumento da deposição de colágeno e do número de vasos sanguíneos, de NAG e de IL-4, quando comparado com os demais tratamentos. Quando as úlceras foram tratadas concomitantemente com LXA4-MS e o antagonista do receptor de LXA4 (ciclosporina H), observamos reversão de cerca de 50% no índice de cicatrização promovido pelo mediador encapsulado, sugerindo que os efeitos tópicos do tratamento com LXA4 são devidos à sua interação com seu receptor específico. Nossos resultados sugerem que novas formulações farmacêuticas para tratamento de úlceras cutâneas poderão ser obtidas com LXA4-MS. / Lipoxin A4 (LXA4) is an eicosanoid derived from the metabolism of arachidonic acid by lipoxygenases (5, 12, and 15-LO), having anti-inflammatory and pro-resolution properties. The strategy of using LXA4 encapsulated in PLGA microparticles as drug is due to some properties that such polymers have to preserve the biological activity of several molecules such as lipids, and promote a prolonged and sustained release of these molecules. In the present study, we hypothesized that the encapsulation of LXA4 in PLGA microparticles (LXA4-MS) preserves its biological activities, and more efficiently accelerates the closing of ulcers induced in the skin of rats. Thus, a model for cutaneous ulcers in the dorsal region of rats was used. The LXA4-MS were fixed in skin using biological adhesive of fibrin, and their effects compared to those induced by empty microparticles (Un-MS), soluble LXA4 or vehicleOur results showed that LXA4-MS accelerate wound healing, once on day 7 after injury, reduced by 80% the diameter of the initial ulcer, while at the same time, the ulcers treated with soluble LXA4, Un-MS or vehicle was decreased of only 60%, 45% and 39%, respectively. The increased healing rate of ulcers induced by treatment with LXA4-MS was accompanied by the decrease of pro-inflammatory cytokines IL-1?, TNF-?, and a TGF-? increasing, an anti-inflammatory cytokine-inducing collagen deposition. In addition, the inflammatory infiltrate was reduced in scar tissue as demonstrated by the decrease of neutrophils, MPO and MMP8 metalloproteinase mRNA. Moreover, LXA4-MS induced increase in collagen deposition and the number of blood vessels when compared with the other controls. When the ulcers were treated concomitantly with LXA4-MS and the antagonist LXA4 receptor (ALX), cyclosporine H, we observed the reversal of about 50% in healing rates promoted by the encapsulated agent, suggesting that topical treatment effects of LXA4 are due to interaction with its own receptor. Our results suggest that new pharmaceutical formulations for the treatment of skin ulcers can be obtained with LXA4-MS.
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Libération contrôlée d'un neuroleptique par voie orale en utilisant des capsules hybrides PLGA-PEG / Alginate/ / Controlled releaseof antipsychotic by oral route using PLGA-PEG/Alginate hybrid capsulesBen Azzouz, Seifeddine 10 November 2017 (has links)
Actuellement les traitements thérapeutiques pour soigner la schizophrénie, par voie intraveineuse ou orale, ne sont qu’en partie efficaces et associés généralement à des effets extrapyramidaux souvent dangereux et très gênants pour les patients. Afin d’augmenter l’efficacité du traitement toute en neutralisant les effets indésirables, ce travail a eu comme objectif de concevoir des capsules composites (PLGA-PEG / alginate) destinées à être administrées par voie orale et capables de libérer localement, de façon spécifique et contrôlée, le neuroleptique halopéridol dans le cerveau. L’optimisation du protocole de synthèse a permis d’obtenir de façon reproductible des nanocapsules de PLGA poreuses monodisperses et peu agrégées, possédant un diamètre hydrodynamique moyen inférieur à 80 nm et une bonne stabilité en solution aqueuse. Une fois fonctionnalisées avec le Poly (éthylène glycol) diamine, des études in vitro ont montré la faible toxicité de ces nanoparticules furtives ainsi que leur capacité à encapsuler une quantité satisfaisante d’halopéridol et de libérer ce principe actif sur une durée d’un mois avec un faible effet « burst ». L’incorporation des nanoparticules pégylées dans des matrices préparées à haute concentration d’alginate et de 100 % CaCl2 a permis d’obtenir des billes nanocomposites possédants une meilleure stabilité à la sortie du milieu gastrique simulé et persistent environ 30 minutes en milieu intestinal simulé. Enfin des études in vivo préliminaires sur des souris adultes utilisant des nanoparticules injectées et des billes nanocomposites ingérées ont démontré l’efficacité de ces systèmes à délivrer l’halopéridol au cerveau. / Currently therapeutic treatments for schizophrenia, intravenously or orally, are only partially effective and generally associated with extrapyramidal effects often dangerous and very troublesome for patients. In order, to increase the treatment efficiency by neutralizing any side effects the aim of this work was to design composite capsules (PLGA-PEG / alginate) intended to be administered by way oral and able to release locally, in a specific and controlled way, the neuroleptic “haloperidol” in the brain. The optimization of the protocol of synthesis allowed to obtain in a reproducible way of the nanocapsules of monodisperse and not very aggregate porous PLGA, having an average hydrodynamic diameter lower than 80 Nm and a good stability in aqueous solution. Once functionalized with Poly (ethylene glycol) diamine, in vitro studies showed the low toxicity of these furtive nanoparticles as well as their ability to encapsulate a satisfactory amount of haloperidol and release this active principle over a period of one month with a low burst effect. The incorporation of the PEGylated nanoparticles in matrices prepared with a high concentration of alginate and 100% CaCl2 made it possible to obtain nanocomposite beads having a better stability at the exit from the simulated gastric medium and persist approximately 30 minutes in simulated intestinal medium. Finally, preliminary in vivo studies on adult mice using injected nanoparticles and ingested nanocomposite balls showed the effectiveness of these systems to deliver haloperidol in the brain.
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Nanopartículas poliméricas e interações com macrófagos / Polymer nanoparticles and interactions with macrophagesBandeira, Anielle Carvalho 16 September 2016 (has links)
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Previous issue date: 2016-09-16 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Nanotechnology involves the creation and use of materials, devices and systems through
control of matter on the manometer scale. It has positive impact on medicine involving the
treatment and diagnosis. Drug delivery systems prepared with nanostructures have the ability
to overcome biological barriers and optimizing drug release. However, one of the main
challenges in the use of these systems is their internalization by macrophages. This study
aims to prepare and characterize polymer nanoparticles formed by biodegradable polymers
and investigate its impact on the function of macrophages in vitro by the observation of cell
viability, phagocytic activity and cytokine production. Also, was investigate the cytokine
profile in serum and splenocytes culture supernatant from Balb/C mice injected with chitosan
nanoparticles. The chitosan and chitosan-containing magnetic nanoparticles were prepared by
ionic gelation crosslinking, resulting in a morphology "nearly spherical" and positive zeta
potential. The PLGA prepared by emulsification and solvent evaporation method presented
spherical morphology and negative zeta potential. All preparations had a diameter smaller
than 300 nanometers and polydispersity index lower than 0.5. Nanoparticles of chitosan and
PLGA affected the viability of macrophages in vitro only at the highest concentration tested (4
mg/mL). The chitosan nanoparticles containing magnetic nanoparticles were internalized by
macrophages after 4h and 24h of incubation. Different nanoparticles triggered high
production of TNF- and low IL12p40 production and IL-10. Serum and splenocytes culture
supernatant was observed in both the production of IFN- as IL-4. However, IL-4 production
was higher than that of IFN-. / A nanotecnologia envolve a criação e utilização de materiais, dispositivos e sistemas através
do controle da matéria na escala manométrica. Apresenta impactos positivos na medicina que
envolve desde o diagnóstico ao tratamento de doenças. Sistemas de entrega de fármacos
apresentam a capacidade de superar barreiras biológicas e otimizar a liberação de um
fármaco. No entanto, um dos principais desafios na utilização desses sistemas é a sua
internalização pelos macrófagos. Esse estudo objetiva preparar e caracterizar nanopartículas
poliméricas formadas por polímeros biodegradáveis e investigar o seu impacto na função dosmacrófagos in vitro através da observação da viabilidade celular, atividade fagocítica e
produção de citocinas. Além do perfil de citocinas no soro e sobrenadante de cultura de
esplenócitos provenientes de camundongos Balb/C que foram injetados com uma suspensão
de nanopartículas de quitosana. As nanopartículas de quitosana e quitosana contendo
magnéticas foram preparadas por gelificação iônica apresentando morfologia reticular e
“quase esférica”, respectivamente, e potencial zeta positivo. As de PLGA preparadas por
emulsificação e evaporação do solvente apresentaram morfologia esférica e potencial zeta
negativo. Todas apresentaram diâmetro menor que 300 nanômetros e índice de polidispersão
menor que 0,5. As nanopartículas de quitosana e de PLGA afetaram a viabilidade de
macrófagos in vitro apenas na maior concentração testada (4mg/mL). As nanopartículas de
quitosana contendo magnética foram internalizadas pelos macrófagos em 4h e 24h de
incubação. As diferentes nanopartículas desencadearam a produção elevada de TNF- e baixa
produção de IL12p40 e IL-10. No soro e no sobrenadante da cultura de esplenócitos foi
observada tanto a produção de INF- quanto de IL-4. No entanto, a produção de IL-4 foi
maior que a de IFN-.
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Avaliação da influência da lipoxina A4 encapsulada em micropartículas de PLGA na cicatrização de úlceras cutâneas em ratos / Influence of Lipoxin A4 encapsulated in PLGA microparticles on wound healing in ratsMouzarllem Barros dos Reis 27 June 2016 (has links)
Lipoxina A4 (LXA4) é um eicosanoide derivado do metabolismo do ácido araquidônico pelas lipoxigenases (5, 12 e 15-LO), tendo propriedades antiinflamatórias e pró-resolução. A estratégia de uso de LXA4 encapsulada em micropartículas de PLGA como fármaco tem fundamento nas propriedades que tais polímeros têm de preservar as atividades biológicas de moléculas diversas, como dos lipídeos, e promover liberação prolongada e sustentada das mesmas. No presente estudo, tivemos como hipótese de trabalho que a encapsulação de LXA4 em micropartículas de PLGA (LXA4-MS) preserva suas atividades biológicas, e mais eficientemente, acelera o fechamento de úlceras induzidas na pele de ratos. Para tanto, um modelo de úlceras cutâneas na região dorsal de ratos foi utilizado. As LXA4-MS foram fixadas em pele com adesivo biológico de fibrina, e seus efeitos comparados com aqueles induzidos por micropartículas vazias (Un-MS), LXA4 solúvel ou veículo (PBS). Nossos resultados mostraram que LXA4-MS aceleram a cicatrização da ferida, pois no 7º dia após a lesão, reduziu em 80% o diâmetro da úlcera inicial, enquanto no mesmo período, nas úlceras tratadas com LXA4 solúvel, Un-MS ou veículo ocorreu diminuição de somente 60%, 45% e 39%, respectivamente. O aumento do índice de cicatrização induzido pelo tratamento das úlceras com LXA4-MS foi acompanhado pela diminuição das citocinas pró-inflamatórias IL- 1?, TNF-?, e aumento de TGF- ?, uma citocina antiinflamatória indutora da deposição de colágeno. Além disso, nas úlceras tratadas com LXA4-MS o infiltrado inflamatório foi reduzido no tecido de cicatrização, como demonstrado pela diminuição de infiltrado celular, de MPO e de mRNA da metaloproteinase MMP8. Por outro lado, LXA4-MS induziu aumento da deposição de colágeno e do número de vasos sanguíneos, de NAG e de IL-4, quando comparado com os demais tratamentos. Quando as úlceras foram tratadas concomitantemente com LXA4-MS e o antagonista do receptor de LXA4 (ciclosporina H), observamos reversão de cerca de 50% no índice de cicatrização promovido pelo mediador encapsulado, sugerindo que os efeitos tópicos do tratamento com LXA4 são devidos à sua interação com seu receptor específico. Nossos resultados sugerem que novas formulações farmacêuticas para tratamento de úlceras cutâneas poderão ser obtidas com LXA4-MS. / Lipoxin A4 (LXA4) is an eicosanoid derived from the metabolism of arachidonic acid by lipoxygenases (5, 12, and 15-LO), having anti-inflammatory and pro-resolution properties. The strategy of using LXA4 encapsulated in PLGA microparticles as drug is due to some properties that such polymers have to preserve the biological activity of several molecules such as lipids, and promote a prolonged and sustained release of these molecules. In the present study, we hypothesized that the encapsulation of LXA4 in PLGA microparticles (LXA4-MS) preserves its biological activities, and more efficiently accelerates the closing of ulcers induced in the skin of rats. Thus, a model for cutaneous ulcers in the dorsal region of rats was used. The LXA4-MS were fixed in skin using biological adhesive of fibrin, and their effects compared to those induced by empty microparticles (Un-MS), soluble LXA4 or vehicleOur results showed that LXA4-MS accelerate wound healing, once on day 7 after injury, reduced by 80% the diameter of the initial ulcer, while at the same time, the ulcers treated with soluble LXA4, Un-MS or vehicle was decreased of only 60%, 45% and 39%, respectively. The increased healing rate of ulcers induced by treatment with LXA4-MS was accompanied by the decrease of pro-inflammatory cytokines IL-1?, TNF-?, and a TGF-? increasing, an anti-inflammatory cytokine-inducing collagen deposition. In addition, the inflammatory infiltrate was reduced in scar tissue as demonstrated by the decrease of neutrophils, MPO and MMP8 metalloproteinase mRNA. Moreover, LXA4-MS induced increase in collagen deposition and the number of blood vessels when compared with the other controls. When the ulcers were treated concomitantly with LXA4-MS and the antagonist LXA4 receptor (ALX), cyclosporine H, we observed the reversal of about 50% in healing rates promoted by the encapsulated agent, suggesting that topical treatment effects of LXA4 are due to interaction with its own receptor. Our results suggest that new pharmaceutical formulations for the treatment of skin ulcers can be obtained with LXA4-MS.
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Efeitos antagônicos da prostaglandina D2 e prostaglandina E2 na resposta imune durante infecção experimental por Histoplasma capsulatum / Opposite effects of prostaglandin D2 and prostaglandin E2 in immune response during experimental infection by Histoplasma capsulatum.Priscilla Aparecida Tartari Pereira 30 October 2013 (has links)
O Histoplasma capsulatum é um fungo dimórfico, patogênico e responsável por graves lesões pulmonares. A infecção é adquirida pela inalação de conídios e posterior conversão para leveduras nos alvéolos e bronquíolos, onde são fagocitadas por macrófagos alveolares residentes e leucócitos que migram para o local da infecção. Recentemente, demonstramos que animais infectados com H. capsulatum e tratados com inibidor da síntese de prostaglandinas apresentaram diminuição de carga fúngica nos pulmões e baço, aumento da produção de nitrito e da fagocitose de leveduras por macrófagos alveolares, e maior sobrevivência, quando comparados com os animais somente infectados. Porém, neste estudo não foram determinados quais subtipos de prostaglandinas participam na patogênese da histoplasmose. Vários grupos de pesquisa têm demonstrado que PGD2 e PGE2 podem ter ações biológicas distintas quanto à remoção de microrganismos no hospedeiro. Desta maneira, é fundamental o entendimento do papel da PGD2 e da PGE2 nos mecanismos efetores dos macrófagos na defesa do hospedeiro, especialmente na histoplasmose. Portanto, o objetivo deste estudo foi investigar a participação da PGD2 e PGE2 na infecção experimental por H. capsulatum. Assim, demonstramos que a PGD2 aumentou a fagocitose e mecanismos microbicidas de macrófagos alveolares infectados in vitro com H. capsulatum. Observamos ainda que a 15dPGJ2, metabólito da PGD2, aumentou somente a fagocitose, e PGE2 inibiu os mecanismos efetores do macrófago. Mostramos ainda o aumento de BLT1 em macrófagos alveolares após adição de PGD2, e a possível ligação desta ao BLT1, e de LTB4 em DP2. Além disso, caracterizamos micropartículas de PLGA contendo PGD2 (MS-PGD2), e investigamos seus efeitos. O tamanho, carga elétrica e morfologia das micropartículas foram adequados para um tratamento intranasal e para fagocitose por macrófagos alveolares. As MS-PGD2 foram fagocitadas e capazes de ativar NF-B, e consequentemente, influenciar na produção de nitrito, IL-1, TNF-, IL-6 e TGF-. Com base nestes dados, avaliamos os efeitos do tratamento da MS-PGD2 ou da MS-PGE2 em animais infectados com H. capsulatum. Estas foram administradas via intranasal em animais infectados e tratados ou não com celecoxibe. Verificamos a diminuição da carga fúngica nos pulmões e baço, diminuição do infiltrador celular no espaço broncoalveolar e de citocinas inflamatórias no pulmão após tratamento com MS-PGD2. Contrariamente, após tratamento da MS-PGE2 observamos maior carga fúngica nos pulmões e baço, e aumento da inflamação no tecido e maior produção de IL-10. Além disso, demonstramos que no 21° dia após infecção, referente ao 7° dia após o término do tratamento com MS-PGD2, a carga fúngica manteve-se reduzida nos pulmões, comprovando assim a eficácia deste tratamento. Posteriormente, utilizando inibidores específicos, HQL-79 e CAY10526, mostramos respectivamente o papel protetor da PGD2 e o deletério da PGE2 na histoplasmose. Em conjunto, nossos dados contribuíram para o entendimento das funções antagônicas da PGD2 e PGE2 nesta micose. / Histoplasma capsulatum is a pathogenic dimorphic fungus and responsible for severe pulmonary lesions. Infection is acquired by inhalation of conidia and posterior conversion to yeasts in the alveoli and bronchioles, in which they are phagocyted by resident alveolar macrophages and leukocytes that migrate to the local infection. Recently, we demonstrate that mice infected by H. capsulatum and treated with inhibitor of prostaglandins synthesis presented a decrease in fungal burden in lungs and spleen, increase in nitrite production and uptake of yeasts by alveolar macrophages, and more survival, when compared with animals only infected. However, in this study, it was not determined what subtypes of prostaglandins participate in pathogenesis of histoplasmosis. Many research groups have demonstrated that PGD2 and PGE2 can have different biological effects regarding to microorganisms elimination in the host. Thus, it is primordial the understanding about the role of PGD2 and PGE2 on effector mechanisms of macrophages in host defense, especially in histoplasmosis. Therefore, the aim of this study was to investigate the role of PGD2 and PGE2 on experimental infection by H. capsulatum. So, we verify that PGD2 increased the uptake and microbicidal mechanisms of alveolar macrophages infected in vitro by H. capsulatum. 15dPGJ2, a PGD2 metabolite, increased only the phagocytosis, and PGE2 inhibited the effector mechanisms of macrophages. Among these results, we showed an increase of BLT1 expression on alveolar macrophages after addition of PGD2, and a possible binding of this mediator to BLT1, and of LTB4 to DP2. Later, as tool of therapeutic investigation, we used PGD2 encapsulation in biodegradable polymer, PLGA, in order to preserve its stability. Size, zeta potential and morphology were adequate for a possible intranasal treatment and uptake by alveolar macrophages. MS-PGD2 were phagocyted and able to activate NF-B, and consequently, to modulate nitrite, IL-1, TNF-, IL-6 and TGF- production. In this context, we purpose a treatment of the infection with MS-PGD2, in comparison to treatment with PGE2. MS-PGD2 were administrated via intranasal in infected mice, treated or not with celecoxib. We verify a decrease of fungal burden in lungs and spleen, less cellular infiltrate and decrease of some inflammatory cytokines. In contrast, after treatment of MS-PGE2, we observed greater fungal burden in the lungs and spleen, and an increase of the tissue inflammation and production of IL-10. Furthermore, we show that on day 21 after infection, referring to the 7th day after the treatment with MS-PGD2, fungal burden remained reduced in the lungs, thus proving the effectiveness of the treatment. Subsequently, using specific inhibitors, HQL-79 and CAY10526, respectively show the protective role of PGD2 and in deleterious to PGE2 in histoplasmosis. Together, our data contribute to the understanding of the antagonistic functions of PGD2 and PGE2 in this mycosis.
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Développement d’une protéine à libération prolongée, mise au point du procédé d’encapsulation sans solvant halogéné et optimisation du profil de libération. / Development of microencapsulation process without toxic solvent, application to sustained protein release.Violet, Fabien 21 September 2015 (has links)
La régénération tissulaire est une voie prometteuse de thérapie dans le cadre des maladies dégénératives. Dans ce but sont conçus les microcarriers pharmacologiquement actifs (PAM). Ce sont des microsphères fournissant un environnement adéquat à la survie et la différenciation de cellules souches par la libération d’un facteur de croissance protéique encapsulé.Pour potentialiser l’intérêt des PAM, les microsphères doivent (1) permettre la libération complète et prolongée de la protéine (2) être formulées sans solvant halogéné par un procédé transposable à l’échelle pilote.Deux stratégies sont menées afin d’améliorer la stabilité et la libération de la protéine. La première consiste à utiliser de nouveaux additifs. Une étude bibliographique révèle le potentiel d’additifs protéiques ; leur application a permis d’augmenter significativement l’activité biologique de la protéine libérée. La seconde stratégie consiste à moduler la matrice de copolymère PLGAP188-PLGA. La modification de ses propriétés physicochimiques (Mw, hydrophobie…) a permis d’accéder à la compréhension de la structure des microsphères et d’obtenir une libération continue.Le développement du procédé de fabrication des microsphères sans solvant toxique associe la technique du prilling avec le glycofurol comme solvant. Cette combinaison se heurte à de nombreux verrous technologiques. La mise au point du procédé a été réalisée à l’aide de plans d’expériences. Ils ont conduit à la production de particules grâce à la modélisation des propriétés physicochimiques du milieu de réception et à la prise en compte des différents paramètres du procédé. / Pharmacologically active microcarriers (PAM) have been developed as innovative tools for tissue regeneration. This microspherical platform provided an environment for the survival and the differentiation of stem cells through the release of encapsulated protein growth factor. To improve the therapeutic efficacy of the PAM, the microspheres have to (1) provide the full and sustained release of the protein (2) be formulated without halogenated solvent by a process with an easy scale-up. The protein release has been studied through two strategies. The first one was to look for a preservation of the biological activity of the protein during the release. A literature review highlighted protein additives. Some of them were incorporated into the microspheres and increased significantly the protein release. The second one was the modulation of the matrix copolymer PLGAP188-PLGA. The modification of its properties (MW,hydrophobicity) permitted to reach a continuous release and to understand the structure of the microspheres. The prilling technique and the use of glycofurol provide an easy transferable process without toxic solvent. Experimental designs were performed to overcome the technological barriers. Through the modeling the physicochemical properties of the reception medium and the study of the process parameters, the formulation has been improved to produce acceptable particles.
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Microparticules à libération controlée : impact du gonflement sur la cinétique de libération de substance active / Controlled release microparticles : impact of swelling on the drug release kineticsGasmi, Hanane 08 December 2015 (has links)
Les études de libération de substance active à partir de système polymériques tels que des microparticules à base d’acide poly(lactique-co-glycolique) (PLGA) ont été largement explorées au cours de ces dernières décennies . L’objectif principal de ce travail consiste à mieux comprendre les mécanismes de transport de masse contrôlant la libération de substance active à partir des microparticules de PLGA. Un nouvel aperçu devait être acquis sur la base de suivi expérimental de la cinétique de gonflement de microparticules. Dans un premier temps, des microparticules à base de PLGA chargées de différents types de substances actives (acide, base et neutre), tels que kétoprofen, prilocaine base libre et dexamethasone ont été préparées par simple émulsion (huile dans eau) en utilisant une méthode d'extraction/évaporation du solvant. Les microparticules obtenues avaient des taux d’efficacité d’encapsulation qui sont variables selon la substance active utilisée. Une caractérisation des propriétés clés des microparticules obtenues a été réalisée en utilisant différentes techniques (microscopie optique, microscopie électronique). La chromatographie par permeation de gel a été utilisé pour déterminer le poids moléculaire du PLGA après exposition des microparticules au milieu de libération à différents temps afin d’évaluer la cinétique de dégradation du polymère. La diffraction des rayons X et la calorimétrie différentielle à balayage étaient utilisés pour étudier l’état physique du polymère, de la substance active pure ainsi que les microparticules chargées en substance active. Les études de libération ont montré deux types de profils de libération : un profil tri-phasique et un profil plus ou moins mono-phasique. Le profil tri-phasique observé est constitué de trois phases : une phase de libération initiale rapide suivie d’une libération constante qui est suivie ; à son tour ; par une seconde phase de libération rapide. En revanche, les différentes phases étaient difficilement distinguées pour le deuxième type de profil obtenu, du fait de la libération rapide de substance active ce qui permet de dire que les profils obtenus étaient plus ou moins mono-phasique. L’élucidation des mécanismes de libération de substance active était basée sur le suivi expérimental de la cinétique de gonflement des microparticules. Comme pour les cinétiques de libération obtenues à partir des microparticules à base de PLGA, différentes phases peuvent être distinguées pour les profils de gonflement. Les transitions d’une phase à une autre semblent s’accorder entre le profil de libération et celui du gonflement. Ainsi, le gonflement des microparticules pourrait contribuer au contrôle de la libération de la substance active à partir des microparticules à base de PLGA. / The drug release studies from polymeric system such as Poly(lactic-co-glycolic) acid (PLGA)-based microparticles have been widely investigated during recent decades. The main objective of this work is to better understand the mass transport mechanisms controlling the drug release kinetics from PLGA microparticles. New insight was to be gained based on the experimental monitoring of the swelling kinetics of single microparticle. Initially, PLGA microparticles containing different type of drugs (acidic, basic and neutral), such as ketoprofen, prilocaine free base and dexamethasone were prepared using simple oil in water emulsion extraction/evaporation solvent technique. The characterization of the key properties of microparticles was performed using different techniques (optical microscopy, electron microscopy). The gel permeation chromatography was used to determine the molecular weight of PLGA following exposure of microparticles to the release medium at various times to assess the kinetic degradation of the polymer. The X-ray diffraction and differential scanning calorimetry were used to study the physical state of the polymer, drug and drug-loaded microparticles. Release studies have shown two types of release profiles: tri-phasic and more or less mono-phasic profile. The tri-phasic profile is composed of three phases: an initial rapid release phase followed by a constant release which is followed by a second phase of rapid release. In contrast, at the investigated higher initial drug loadings, different release phases could hardly be distinguished: The profiles were more or less mono-phasic. The elucidation of drug release mechanisms was based on the experimental results of the swelling kinetics of single microparticles. As for drug release, distinct phases can be distinguished for microparticles swelling. The transition from one phase to another seem to coincide for microparticle swelling and drug release. Thus also microparticle swelling might contribute to a significant extent to the control of drug release.
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Implants formés in-situ pour le traitement des poches parodontales : évaluation in-vitro et in-vivo / In-situ forming implants for periodontal pockets treatment : in vitro and in vivo evaluationAgossa, Kevimy 27 November 2018 (has links)
La frequence elevee et les consequences considerables des maladies parodontales sur la qualite de vie orale et la sante generale font de ces pathologies une preoccupation de sante publique. Le developpement de traitements innovants est un moteur essentiel du progres dans la prise en charge de ces maladies. Ce travail s’interesse a l’utilisation d’agents antimicrobiens (non-antibiotiques) et anti-inflammatoires comme adjuvants au debridement mecanique des poches parodontales. Il est consacre a la mise au point et a l’optimisation d’une forme galenique appelee implant forme in-situ (IFIS), a base de polymeres biocompatibles et resorbables, concue pour le traitement des poches parodontales. Ce dispositif est injecte dans la lesion ou il libere de facon prolongee une combinaison d’antiseptique (chlorhexidine) et d’anti-inflammatoire (ibuprofene). Nos resultats montrent (i) in-vitro la superiorite de l’IFIS en termes de proprietes mecaniques et antimicrobiennes sur des systemes actuellement commercialises pour le meme usage et (ii) in-vivo, chez l’animal, un effet positif de l’IFIS sur la cicatrisation parodontale. Cette etude valide l’usage d’IFIS a base d’acide Poly(D,L-lactique-co-glycolique) (PLGA) comme support de liberation controlee de principes actifs dans la poche parodontale. Elle montre egalement que la combinaison d’un antiseptique et d’un anti-inflammatoire au sein du meme systeme de liberation locale pourrait ameliorer le traitement des parodontites tout en limitant le recours aux antibiotiques. / Periodontal diseases are highly prevalent oral conditions which strongly impact the oral quality of life and general health. Progress in the management of periodontal diseases depends, at least partly, on the development of novative and effective periodontal treatments. This work is concerned with the use of non-antibiotic antimicrobials and antiinflammatory drugs as adjunctive periodontal therapy. The aim was to develop a novel polymer-based, biocompatible and resorbable local drug delivery system (in-situ forming implants -ISFI-), which could be injected into periodontal pockets and control the delivery of both an antiseptic (chlorhexidine) and an anti-inflammatory agent (Ibuprofen). The physical key properties and antimicrobial activity of the proposed implants were very promising in vitro suggesting an improvement as compared to currently marketed drug delivery systems for periodontitis treatment. In vivo results are consistent with the latters, showing a positive effect of ISFI on periodontal wound healing in an experimental mice model of periodontitis. Data from this study support Poly(D,L-lactic-co-glycolic) acid (PLGA) as an attractive formulation for local drug-delivery into periodontal pockets. Our results further suggest that the local delivery of both an antiseptic and an anti-inflammatory drug is a promising adjunctive treatment of periodontitis. This should additionnaly reduce the use of systemic antibiotics in periodontal treatment and contribute to the combat against the development of bacterial resistances.
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